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Trypanosoma brucei contains an ATP-dependent phosphofructokinase (PFK), located in its glycosomes, which are peroxisome-like organelles sequestering the majority of its glycolytic enzymes. In this paper, we report the cloning and sequencing of the single-copy gene encoding this enzyme. Its amino-acid sequence is more similar to pyrophosphate (PPi)-dependent PFKs than to other ATP-dependent PFKs. a phylogenetic analysis suggests that the enzyme must have been derived from a PPi-dependent ancestral PFK, which changed its phospho-donor specificity during evolution. The enzyme is no longer capable of using PPi as phospho substrate, nor can it catalyze the reverse reaction as PPi-PFKs generally can. Moreover, the presence of a high pyrophosphatase activity in the cell renders it unlikely that PPi can function as free-energy source in present-day trypanosomes. It remains to be determined which mutations were responsible for the chage in phospho-substrate specificity of the trypanosomatid PFK. As a result of its particular evolutionary hitory, the T. brucei PFK shows many structural differences, even at the active site, when compared with other ATP-dependent PFKs. These differences offer great potential for the structure-based design of trypanocidal drugs. [ABSTRACT FROM AUTHOR]

The phosphofructokinase of the red cells of rats is exquisitely sensitive to inhibition by ATP. The apparent Ki for ATP is decreased with lower pH values and fructose-6-phosphate concentrations. 2,3-Bisphophoglycerate, which is present in the red cells of man and many mammals, inhibits the phosphofructokinase in a synergistic manner with ATP. The inhibitory effect of phosphoenolpyruvate is biologically insignificant. It may be estimated that the enzyme would be practically completely inactive under cellular conditions without the action of positive effectors. There exist a multitude of positive effectors. Significant activations under cellular conditions are exercised by AMP, glucose 1,6-bisphosphate and inorganic phosphate. Fructose 1,6-bisphosphate activates to a limited extent and ADP only at alkaline pH values. All positive effectors produce a decreased inhibition by ATP and increased affinity of the enzyme for fructose 6-phosphate. AMP is the activator with the highest affinity, while glucose 1,6-bisphosphate may be considered as a baseline activator owing to its sluggish turnover. The data may be fitted in a qualitative manner to the model of Monod, Wyman and Changeux. It is possible that the negative anionic effectors act on the same site. [ABSTRACT FROM AUTHOR]

Rabbit muscle phosphofructokinase uniformly carboxymethylated with iodo[2-14C]acetate consists of subunits with a molecular weight of 80000 ± 5000. The subunit polypeptide chain contains 16 and 52 residues respectively of cysteine and arginine and, contrary to previous results, peptide mapping experiments gave no indication that phosphofructokinase chains yield fewer than the expected numbers of cysteine and arginine containing peptides. To test further for the possible occurrence of repeat sequences within a single subunit chain, cysteine-containing peptides were isolated and sequenced from tryptic and thermolytic digests of s-[2-14C]carboxymethylated phosphofructokinase. In all, 15 different cysteine sequences (comprising a total of 104 residues) were identified, showing that not more than one of an expected 16 cysteine-containing sequences is repeated, and that the subunits in phosphofructokinase are of unique sequence along their entire length. The near quantitative isolation of several cysteine-containing peptides shows further that all subunits are of similar if not identical sequence. [ABSTRACT FROM AUTHOR]

Treatment of liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase with the arginine- specific reagent, phenylglyoxal, irreversibly inactivated both 6-phospbofructo-2-kinase and fructose- 6-bisphosphatase in a time-dependent and dose-dependent manner. Fructose 6-phosphate protected against 2,6-phosphofructo-2-kinase inactivation, whereas MgGTP protected against fructose-2,6- bisphosphatase inactivation. Semi-logarithmic plots of the time course of inactivation by different phenylglyoxal concentrations were non-linear, suggesting that more than one arginine residue was modified. The stoichiometry of phenylglyoxal incorporation indicated that at least 2 tool/tool enzyme subunit were incorporated. Enzyme which had been phosphorylated by cyclic.AMP-dependent protein kinase was inactivated to a lesser degree by phenylglyoxal, suggesting that the serine residue (Ser32) phosphorylated by cyclic-AMP-dependent protein kinase interacts with a modified arginine residue. Chymotryptic cleavage of the modified protein and microsequencing showed that Arg225, in the 6-phosphofructo-2-kinase domain, was one of the residues modified by phenylglyoxal. The protection by fructose 6-phosphate against the labelling of chymotryptic fragments containing Arg225, suggests that this residue is involved in fructose 6-phosphate binding in the 6-phosphofructo-2-kinase domain of the bifunctional enzyme. [ABSTRACT FROM AUTHOR]

A 5S-rRNA-containing ribonucleoprotein was purified to homogeneity from a rabbit muscle extract through its affinity to phosphofructokinase-1 and then stucturally characterized. This RNP was compared to the 5S- rRNA-containing ribonucleoprotein extracted from rabbit liver ribosomal 60S subunits with EDTA. Analytical gel filtration revealed a molecular mass of 70-80 kDa for both complexes. Gel electrophoresis of the ribosomal complex revealed three protein components, one migrating as a band of 35 kDa and two other small polypeptides of apparently 16.5 kDa and 17.5 kDa. In the sarcoplasmic RNP these small polypeptides were absent. However. besides a major component of 35 kDa, up to five slightly larger and smaller species of 31.5-36.5 kDa were detected. Despite this heterogeneity, only one N-terminal amino acid sequence was obtained for the isolated sarcoplasmic protein, suggesting a C-terminal heterogeneity of one single polypeptide. Within the first 46 amino acid residues no difference between the sequences of the isolated 35-kDa components of sarcoplasmic and ribosomal complexes was found. Homology criteria indicated that this component belongs to the ribosomal protein L5 family. The RNA was identified by complete enzymatic sequencing as 5S rRNA; it was also identical in both complexes and is strongly homologous to 5S rRNA of man. Both L5 - 5S-RNA complexes could be resolved by hydroxyapatite chromatography into three species still consisting of both protein and RNA. 5'-Terminal dephosphorylation experiments showed that this heterogeneity is exclusively due to the differing number (1 -3) of 5-terminal phosphates. The two additional low-molecular-mass proteins were stably associated to the ribosomal RNP at high salt concentrations in a stoichiometry of about 2:1. They were identified as the acidic phosphoproteins P2/β by N-terminal sequencing. High phosphate concentrations facilitated their dissociation from the L5 - 5S-RNA complex. For the sarcoplasmic L5 - 5S-RNA complex a hitherto unknown interaction with phosphofructokinase-1, affecting the enzymatic properties, was demonstrated. [ABSTRACT FROM AUTHOR]